This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We will optimize a method for rapid identification of protein kinase substrates. In this method a protein kinase is first engineered to accept an ATP analog that allows it to uniquely label its substrate with a bio-orthogonal phosphate analog. Then we will use a highly specific covalent capture-and-release methodology to rapidly purify tagged peptides derived from labeled substrate proteins. Initial work will focus on the optimization of this technique, followed by application to several kinases with known mitochondrial targets.